1. Field of the Invention
The present invention generally relates to genetic changes associated with the onset of sporadic breast cancer, and more particularly to methods and compositions for identifying such changes and for screening women at risk of developing the disease.
2. Description of Related Art
The number of breast cancer cases diagnosed each year worldwide is about one million (1). Breast cancer represents 18% of all cancers in women and, with the exception of skin cancer, is the most common site of cancer in women. In decreasing order, the incidence of other cancers in women are cervix, colon/rectum, stomach, endometrium, lung, ovary, mouth/pharynx, esophagus and lymphoma (1). The fact that a similar pattern is widespread throughout the Western world suggests that related genetic changes may be the origin of cancer development at these sites.
Many risk factors have been defined for breast cancer (1-10). When specific risks are given relative ranks, an interesting pattern emerges. The major four risk factors are age (relative risk 10) followed by geographic location, previous breast cancer, and previous benign breast disease (relative risks of 4 to 5) (1). Reproductive history is next most important with relative risks of 2 to 3 (1). Other factors such as diet, alcohol consumption, socioeconomic group and family history are in the relative risk range of 1.3 to 2 (1). Notably, the risk of oral contraceptives or hormone replacement therapy is low, in the relative range of 1.2 to 1.4 (1). From these data, it can be concluded that the origin of breast cancer, and hence the number of causative genes, are yet to be identified. While modification of personal habits, reproductive considerations, and behavior can reduce risk, the benefits are modest and certainly offer no guarantees. Thus far, traditional epidemiology has not provided “the” origin of breast cancer. Indeed, based on epidemiological data, it has even been suggested that breast cancer arises from a single cause (11). But this conclusion came with the statement that the cause was still unknown. With the growing importance of genetic analysis, it now appears likely that epidemiology will move into this arena to make further advances.
Other investigators have made observations that are critical to understanding the genetic origins of breast cancer. First, it is clear that only a small minority of cancers originate from germ-line mutations (12). Second, there are more than 100 changes in gene expression detectable in breast cancer cells versus normal breast epithelium (12). This large number promises to increase to 200 to 300 with new technology (13,14). Based on a recent discussion (12), cancer mutations have been defined as Class I, which involve changes in gene DNA sequences, and Class II genetic alterations, in which changes in gene expression are detected by mRNA analysis (12). By selecting specific RNA species for further study, information can be gained, but there is no assurance which change(s) is causative. Indeed, it seems highly unlikely that cancer development requires this huge number of gene alterations. It is more likely that most of the changes are the result of malignant transformation, not the cause. It may be possible to use mass gene expression analysis (e.g. microarray technology) to predict breast cancer risk or susceptibility, but for now this seems distant.
It seems reasonable to retreat from these types of “shotgun” analysis and approach the issue from another perspective. In this proposal, focus is placed on genetic changes that are more subtle and are represented by loss of heterozygosity (LOH) or other allelic imbalances (AI). These genetic alterations are known to be associated with a high risk of cancer development. There are categories of women with three or more times higher risk of developing breast cancer than average. They are often classified into a group termed “familial breast cancer”. They have (i) a first degree relative with breast or ovarian cancer, (ii) one first degree relative with disease diagnosed under the age of 40, (iii) two first or second degree relatives with breast or ovarian cancer under the age of 60, or (iv) three first or second degree relatives with breast or ovarian cancer on the same side of the family (1). It is tempting to conclude that this represents an inherited trait or at least a propensity to development of the disease. This conclusion must be tempered however by an understanding that “inherited” might include as yet unrecognized non-biological inheritance such as culture inheritance and common environmental conditions (15). While genetic predisposition remains a strong possibility, it may not be of the type seem with BRCA1 (16) and BRCA2 (17) which are inherited as autosomal dominants from either parent, albeit with varying penetrance. Equally, it is possible that the predisposition is a recessive inheritance, also with varying penetrance, or a recessive mutation that leads to breast cancer development. It may also represent inherited or acquired genetic abnormalities such as loss of heterozygosity (LOH) or other allelic imbalances (AI) such as abnormal gene numbers.
A familial aspect to breast cancer has been recognized for some time. However, this term may have different meanings. It might indicate “familial clustering” which is the existence of several cases in an extended family. Because breast cancer will occur in 10% of women, chance alone will allow some familial clusters depending upon the number of females in a related group. Therefore, because a family has more than one member with the disease, it does not necessarily follow that there is a genetic cause. In contrast, the familial aspect of breast cancer might also indicate “familial aggregation” which is increased risk to close relatives of women with breast cancer compared to relatives of women without the disease. Familial aggregation is another matter. Its existence may depend on genetic and/or non-genetic causes. As discussed above, the presence of breast cancer in one or more first degree relatives increases risk significantly. As the number of breast cancers in first and second degree relatives increases, especially at younger ages, there is strong reason to hold that there are familial factors that may be genetic. If genetic in origin, identification of these genes will be a major advance in understanding breast cancer (18). Genes which show LOH or AI are strong candidates for identification. The composition and methods of identification of such genes is a primary focus of this disclosure.
The question is: “What genes are being sought to explain familial aggregation”? The low frequency of BRCA1 and BRCA2 does not support the view that they account for one million new cases each year worldwide (18). Although somewhat difficult to assess, one report indicates an estimated frequency of significant mutations in these two genes in the US and UK of 0.0005 to 0.002 (19). Another report (25) states mutations in 1 in 152 and 1 in 833 for mutations in these two genes. It is higher in some regions of the world such as Scandinavia and approaches 1 in 40 in Ashkenazi Jews (20). The major issues resulting from the lack of broad application of BRCA1 and BRCA2 are what other genes are to be sought and by what methods.
There are three separate genetic syndromes that are associated with above average rates of breast cancer development (26). These are Li-Fraumeni Syndrome, Cowden's Syndrome and Ataxia Telangiectasia (AT). Each appears to involve a different gene lesion or small set of lesions, but nonetheless all contribute to a higher risk of breast cancer. These syndromes illustrate the point that a small number of critical mutations are likely involved in breast cancer development. In addition, Lynch Syndrome II is associated with breast cancer development (28). The genetic lesion is important in Lynch Syndrome because it involves impairment of the critical process of DNA mismatch repair (29,30).
Li-Fraumeni Syndrome (LFS) is a very rare germ-line mutation in p53 (31) that increases premenopausal breast cancer as well as sarcomas, brain tumors, leukemia and adrenocortical cancer (32,33). LFS is an autosomal dominant syndrome for which genetic testing is not yet available. It is thought that availability would not change medical management (34). Nonetheless, p53 mutations occur in 30% of sporadic breast cancers (27). The very diverse types of p53 mutations in breast cancer pose a problem for genetic testing (27). One study suggests that mutations at codon 248 might be associated with higher breast cancer risks (35). While screening for P53 changes alone may not be productive, combination screening of P53 with other genes may be very informative based on the accepted view that cancer development is a multistep process involving a relatively few genes.
Cowden's Syndrome is characterized by excess breast cancer, gastrointestinal malignancies, thyroid disease, and other benign conditions (36). Cowden's syndrome carries a lifetime breast cancer risk of 25 to 50%. There is usually early onset and often appearance of bilateral disease (37). There is a germ-line mutation in PTEN, a protein tyrosine phosphatase with homology to tensin. PTEN acts as a tumor suppressor and functions in the control of the cell cycle (38). As with P53, screening for PTEN mutations alone may not be informative, but in combination with other genes, may provide important prognostic value.
Ataxia Telangiectasia (AT) is an autosomal recessive disorder characterized by neurologic deterioration, telangiectasias, immunodeficiencies, and marked hypersensitivity to ionizing radiation. Approximately 1% of the population may be heterozygote carriers of the AT mutation (ATM). Over 200 mutations have been identified in the ATM. The majority are truncations (39). ATM proteins have a role in cell cycle control (40). ATM individuals are susceptible to cancer . Studies have shown that even heterozygotes are at elevated risk for breast cancer (41,42). This is the case despite the fact that the ATM is not identified in breast cancer specimens in excess of its occurrence in control populations (43-45). The link between ATM and breast cancer may be related to the kinase coded for by the AT gene. Its absence leads to chromosomal instability, a condition often associated with breast cancer. Because of the relative high frequency of heterozygotes in the U.S. and European populations, analysis of the AT gene plus other genes will be useful.
Lynch Syndrome II is a form of several related diseases first reported as “cancer family syndrome” (46). Other types of this disease are Lynch Syndrome I, (also called hereditary nonpolyposis colon cancer—HNPCC), Miur's Syndrome (also called Torre's Syndrome), Down family Syndrome, Bloom's Syndrome and finally Dyskeratosis congenital (46). For the purposes of example, Lynch Syndrome II will be discussed. This discussion is intended to encompass these other related forms of the disease as they relate to breast cancer development. Lynch Syndrome II is accompanied by the aggregation of colon, endometrial, ovarian and breast cancer in families (28). This disease is due to mutations in DNA mismatch repair genes designated hMSH2 and hMLH1 (47). The result of these mutations is to create microsatellite instability (48). Microsatellite instability is used today to measure the mutations in populations of specimens from colon cancer patients (49). This has given a range of estimates of the size of population bearing these mutations. It seems likely that the two mutations lead to the accumulation of mutations throughout the genome. With time, genes important in growth regulation of mucosal cells become altered and result in the onset of cancer.
Cancer is now generally thought to be a multistep disease that arises in response to genetic changes altering key regulatory proteins within the cells. The mutations leading to cancer can be present in the germ line or can arise as somatic mutations in the tissues. It is clear that a progression exists whereby normal cells change to arrive at the fully malignant state capable of metastasizing to distant body sites. While many gene expression changes can be detected by sophisticated technology, it is reasonable to conclude that the powerful methods applied mask the fact that only a relative few changes ultimately result in cancer.
Although advancement has been made toward understanding genetic predisposition to development of certain breast cancers, there remains a pressing need for ways to identify the genetic changes associated with the onset of sporadic breast cancers, which represent about 6070% of the total number of cases diagnosed each year that have no known genetic origin. There is also a great need for ways to screen individuals for risk of developing the disease and for taking appropriate preventative measures.